One of the new emerging techniques used today in the research of molecular biology and genetics is fluorescent labeling of a biological specimen. According to this technique, fluorescent probes are used to mark the specific locations in a biological specimen aimed at detecting different genes, chromosomes, DNA strands, proteins, and bacteria.
In recent years, the fluorescent labeling based techniques have started to push their way into the diagnostic world, and it is anticipated that in the near future diagnostic assays based on fluorescent labeling will be used more and more routinely.
According to conventional techniques, the detection of fluorescent probes is done in research laboratories by using an “off the shelf” fluorescent microscope. The use of a fluorescent microscope was a logical choice, since this machine was readily available in most research labs. Furthermore, it was a familiar tool to all researchers, and had the benefit of being a multi purpose platform used for other lab applications as well.
However, the detection of fluorescent probes in a biological sample by means of the conventional fluorescent microscope suffers from several drawbacks associated with the following. Today, in diagnostic laboratories that use fluorescent techniques, an operator with genetic training typically manually operates a fluorescent microscope. The operator must manually select the correct objective and filters, manually scan the slide and search for good genetic material, focus on each image, analyze the fluorescent signals, and write down his analysis. The operator has to look through a binocular eyepiece during the entire process, which is a cumbersome and tiring process. Thus, an operator cannot work on the microscope for more than a few hours continuously, and not more than 8-10 hours daily. This of course limits the number of tests a lab can perform, thus limiting the lab's throughput significantly.
Furthermore, the laboratory, where this analysis is done, has to be in blackout conditions. This is associated with one of the major problems of using fluorescent labeling for routine diagnostic assays, consisting of keeping the fluorescent labeling “alive” long enough to finish the entire procedure, which typically includes scanning the sample on a slide, looking for region of interests (ROIs) in the sample (for example, a nucleus of the cell or a chromosome), focusing on the ROIs, taking an image thereof, refocusing on sub areas within the ROI (for example labeled genes), and taking images of the these sub areas as well. This procedure takes quite a while, since a large number of ROIs must be considered to achieve the high reliability required from an assay used for diagnostic purposes. For example, in prenatal FISH tests (fluorescence in situ hybridization) at least 100 good regions of interest (nuclei) are needed to be imaged for giving a reliable diagnosis from the test (“Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH)”, Prenat Diagn 2001; 21: 293-301. DOI: 10.1002/p. 57). FISH method is typically used to detect the absence or excess of a specific gene (e.g., elastin gene) from a chromosome, e.g., to detect the presence of down syndrome.
To detect 100 good enough regions of interest, one must scan several hundreds of fields on the sample. Working for so long on the sample raises the problem of bleaching. Bleaching of a sample causes the fluorescent probes to fade, thus making the reading of the sample impossible. This phenomenon, which occurs within minutes, is stimulated by light and oxygen. Operation with the conventional fluorescent microscope thus requires operation in the dark, and implies that other activities requiring light cannot be carried out at the same time and place, when fluorescent analysis is in process. As a result, all laboratory work has to be halted when fluorescent signals are analyzed, or a separate room has to be assigned for the fluorescent microscope. Furthermore, the necessity to work in a dark environment, affects the performance of the microscope operator. Working in the dark, is no doubt, a cumbersome task.
Other environmental hazards of the conventional techniques, such as heat, humidity, radiation, electromagnetic waves, also have undesired influence on some biological samples. With the conventional microscope and conventional technique, operating personnel are exposed to safety hazards due to UV light typically used to excite the fluorescent sample, but is harmful to people.
The use of a “semi-automatic” fluorescent microscope set-up has been proposed (BX51 Epi-Fluorescence Microscope commercially available from Olympus). In this set-up, a digital camera and a computer are added to the fluorescent microscope solely for archiving the images so as to enable reviewing the images at a later time. An “automatic” fluorescent microscope set-up (DM RXA2 Fluorescence Microscope commercially available from Leica) allows for integrating the “off-the-shelf” components such as a microscope, digital camera, scanning stage, and computer, in conjunction with a software package that controls the operation of these components. However, using the “off-the-shelf” components that were not designed specifically for fluorescent diagnostic tasks (to comply with the demands of the fluorescent-based diagnostic world) obviously decreases performance and increases costs of the optical system.